Chemically selective imaging platforms are important for monitoring biological processes such as drug penetration into tissues and metabolite conversion in live cells. Light can be used in such platforms since electromagnetic radiation is modified in various ways when it passes through a substance or reflects off the substance. Various compounds and other chemical structures, such as atomic bonds, can be detected by locating absorption bands in the light from the substance. Raman scattering is an effect in which light of an incident wavelength strikes an object and the object radiates light of a slightly different wavelength. The difference between the wavelengths is correlated with the energy of an electron band in the object.
Bio-analytical measurement techniques, including Raman spectroscopy, NMR spectroscopy and mass spectroscopy, are widely used for the molecular detection, further leading to deeper research in histology and cell mechanisms. These and other schemes analyze the spectra of complex objects. For example, confocal-fluorescence spectral imaging provides two-dimensional images of an object, e.g., a cell, at each of a number of wavelengths. However, these sets of images can be time-consuming to acquire, e.g., five seconds per wavelength for some techniques. This limits the usefulness of these techniques for live cells, which can move during imaging.
Moreover, many prior schemes are limited to the single point detection without spatial distribution of molecules. There is thus a need for high resolution imaging tools combined with molecular mapping ability. Raman-scattering-based spectroscopic imaging has found wide use in biomedical, pharmaceutical, and material sciences due to its high chemical selectivity and noninvasive nature. However, because spontaneous Raman scattering is a feeble effect, the imaging speed of Raman microscope is limited to hundreds of milliseconds per pixel, or tens of seconds per frame. Such slow data acquisition speed inhibits the application of Raman microscopy to live imaging or large-area mapping. For example, confocal Raman microscopes that provide sub-micron resolution and selective-chemical mapping have been used, but such microscopes have a low scattering cross section, especially to dynamic systems.
Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy is a scheme that provides a spectrum at each pixel. The CARS signal is generated at a frequency different from the frequency of the incident light, so multiplex CARS detection is straightforward using a sensitive charge coupled device. Multiplex CARS microscopy based on broadband excitation and parallel spectral detection has been demonstrated with the pixel dwell time as short as 20 ms. However, this speed does not permit imaging of a highly dynamic living system. Moreover, the CARS signal contains a non-resonant background that makes quantitative analysis difficult. Hyperspectral CARS or SRS microscopy has been demonstrated by spectral scanning of a narrowband laser and collection of images at a series of Raman shifts. Nevertheless, this approach is not applicable to living systems due to spectral distortion caused by dynamics inside live cells during the period of spectral scanning. Moreover, CARS can have difficulty detecting chemical bonds such as a C—H bond.
The Stimulated Raman Scattering (SRS) process has recently been employed for high-speed vibrational imaging. SRS provides strong Raman signal and exhibit no non-resonant background. SRS is a third order nonlinear optical process, which involves two laser fields, namely a pump field at ωp and a Stokes field at ωS. When the beating frequency (ωp−ωS) is tuned to excite a molecular vibration, the energy difference between ωp and ωS pumps the molecule from a ground state to a vibrationally excited state. The laser field manifests this as a weak decrease of pump beam intensity, called stimulated Raman loss (SRL), and corresponding increase of Stokes beam intensity, called stimulated Raman gain (SRG). Using heterodyne detection, SRS is able to offer quantitative spectral information with a pixel dwell time of few μs.
FIG. 6 shows an example of SRS measurement of a sample according to conventional schemes. The angular frequency ωp of a narrowband pump beam is scanned and the angular frequency ωs of a narrowband Stokes beam is held fixed. With ωp−ωS tuned to a molecular vibration at frequency Ωn, nε[1, 2, 3], the pump beam intensity is slightly decreased by stimulated Raman loss (SRL; ΔIP) and the Stokes beam intensity is slightly increased by stimulated Raman gain (SRG; ΔIS). Only parts of the spectrum at which SRL occurs are illustrated here. Dashed lines show the incident radiation before interaction with the sample; solid lines show the radiation resulting from interaction with the sample.
To measure the weak laser intensity change ΔIp, e.g., on the order of 0.01% or smaller, a heterodyne detection approach has been used. In the case of SRL, the Stokes beam intensity IS is modulated and the pump beam intensity Ip is recorded by a photodiode. The induced modulation is then extracted by a lock-in amplifier. Theoretically, the modulation depth induced by SRL, ISRL/Ip, is linearly proportional to the Raman cross section, σ, molar concentration of the target molecule, N, and the Stokes beam intensity, i.e.,ISRI/Ip∝σNIS.  (1)A megahertz (MHz) modulation rate can be used to reduce effects of low frequency laser noise. Lock-in amplifiers (analog or digital) are commonly used for extraction of heterodyne-detected signals like SRS. So far, fast SRS imaging is mostly implemented by narrowband laser excitation of single isolated Raman band. Single-color SRS is, however, not able to resolve overlapping Raman bands contributed by target molecules and background tissue components. Moreover, the imaging of living systems using SRS is made more difficult by the movement of objects during the wavelength tuning. Multi-color SRS imaging has been demonstrated by using three lock-in amplifiers. Such a scheme is, however, impractical for acquisition of a complete Raman spectrum due to high cost of radio-frequency (RF) lock-in amplifiers.
There is, therefore, a need of improved ways of collecting spectral data of samples such as objects, tissue, or micro-organisms, and particularly of collecting spectral data over areas of such samples.
Reference is made to WO2013/110023, International Application No. PCT/US2013/022348, U.S. Pat. No. 6,809,814, and U.S. Pat. No. 6,108,081, each of which is incorporated herein by reference.